PROJECT SUMMARY/ABSTRACT The interference of DNA polymerase movement by DNA damage induced by endogenous, environmental, and chemotherapeutic agents is both a cause of cancer and aging in humans and a common mechanism of action of many anti-cancer drugs. This genotoxin-associated replication stress is also a well- recognized activator of the ATR (ataxia telangiectasia-mutated and rad3-related) protein kinase, which plays critical roles in regulating DNA replication and cell cycle phase transitions during the cellular DNA damage response (DDR). Small molecule inhibitors of ATR have emerged as potential adjuvants to improve the effectiveness of common cancer chemotherapy regimens. Unfortunately, our understanding of the role of ATR in the DDR may be biased towards cellular processes involving DNA synthesis and cell division because of the model systems of actively replicating cells that are typically used to study ATR function. However, using non- replicating cultured cells in vitro and human skin tissue explants ex vivo, our preliminary data have revealed a novel mode of ATR activation that is closely linked with transcription stress and specifically with the XPB subunit of the multi-functional protein TFIIH (transcription factor II-H). Moreover, we have found that in striking contrast to the DNA damage-sensitizing effects of ATR kinase inhibition on replicating cells, ATR inhibition in non- replicating cells instead protects non-replicating cells from the lethal effects of several DNA damaging agents. The objective of this proposal is to therefore more clearly define the mechanisms of ATR kinase activation and function in non-replicating human cells and to determine whether ATR inhibitors provide therapeutic benefit to important cell populations of human tissues exposed to DNA damaging agents. The central hypothesis of this proposal is that the mechanism of ATR kinase activation in non-replicating cells exhibits unique properties in comparison to replicating cells and that transcription-associated ATR kinase signaling has profoundly different effects on cell and tissue fate in response to DNA damage. The rationale for this proposed research is that it will provide a more complete understanding of how the ATR kinase impacts cellular and tissue responses to DNA damaging agents in humans and may lead to the use of ATR kinase inhibitors to limit the toxicity of certain DNA damaging compounds. Our hypothesis will be tested by carrying out the following three specific aims: 1) Define the mechanism of ATR kinase activation in non-replicating quiescent and differentiated human cells exposed to genotoxic stress; 2) Characterize the positive and negative consequences of ATR kinase inhibition in non- replicating cells in vitro; and 3) Validate the modes of ATR kinase activation and function in non-replicating cells of human and mouse skin tissue ex vivo and in vivo. Our approach is innovative because it will investigate an unexplored and physiologically relevant aspect of the DNA damage response in human cells and tissues. The proposed research is significant because it will provide novel mechanistic insights into how modulation of ATR- dependent DNA damage signaling may provide therapeutic benefits in human cells and tissues.